HspA1A, a 70‐kDa heat‐shock protein, contains several distinct lipid‐binding sites

Abstract

HspA1A, a stress‐inducible seventy‐kilodalton heat shock protein (Hsp70), is a molecular chaperone that plays critical roles in protein homeostasis and cell survival. In addition to its functions in protein homeostasis, HspA1A also functions at different cellular membranes in stressed and cancer cells, where it binds to lipids, including phosphatidylserine (PS) and Bis(Monoacylglycero)Phosphate (BMP). Although the interactions between HspA1A and lipids have important biological consequences, their mechanistic details remain elusive and unexplored because the amino acids responsible for the lipid binding remain largely uncharacterized and the relationship between the chaperone and lipid‐binding functions remains unknown. To clarify the mechanism of the HspA1A‐lipid interactions, we first characterized the properties of a mutation from Tryptophan to Phenylalanine (W90F), known to affect binding of HspA1A to BMP and a Lysine to Alanine (K71A) mutation, which results in complete loss of the chaperone function. Experiments using the lipid vesicle sedimentation (LVS) method and Surface Plasmon Resonance revealed that although the W90F‐HspA1A binding to BMP was different from the WT, this mutation did not affect the binding to PS. Furthermore, our results revealed that the K71A mutation did not significantly change the binding of HspA1A to lipids under any of the conditions tested. These results allowed us to make two predictions: first, the lipid‐binding sites for PS and BMP are lipid‐specific, and second, the chaperone and lipid‐binding functions are distinct. To support the first prediction, we used a combination of structural superimpositions, sequence alignments, and literature observations and predicted three putative lipid‐binding regions on the HspA1A molecule. We tested these predictions using the LVS assay for 20 single or double mutations spanning two of these regions. Our results revealed that only two of these mutations affected the binding to PS, while a third one altered the binding to BMP. To test the second prediction, we performed a series of experiments to determine the effect of lipid binding to the secondary structure and chaperone functions of HspA1A. We used Circular dichroism spectrometry, measured the release of inorganic phosphate, and determined the rate of refolding of chemically denatured beta‐galactosidase in the presence or absence of particular lipid. These experiments revealed that lipid binding did not alter the secondary structure of the protein and affected neither the rate of ATP hydrolysis nor the rate of protein refolding. Together our findings provide further evidence that HspA1A binds to PS and BMP using different amino acid sites and support the notion that the chaperone and lipid‐binding functions of HspA1A do not overlap. These findings provide the basis for future experiments to test the effects of these mutations on the membrane‐localized functions of HspA1A in cancer and stressed cells.